Genome-wide association studies (GWAS) of disease risk serve as a promising strategy to identify novel therapeutic targets. However, going from SNP association to a high-throughput screen (HTS) to identify novel drugs requires many steps and has not yet been successfully accomplished. Our team has exciting preliminary data demonstrating that HTS assays can be developed from GWAS of complex traits. We will perform one of the first HTS based on GWAS findings - a confirmed association of a common allele at the CD40 locus and risk of a common autoimmune disease, rheumatoid arthritis (RA). We will identify novel intracellular inhibitors of CD40-mediated NF-kB signaling in B cells, which can ultimately be used to treat RA. Although others have searched for inhibitors of NF-kB signaling, and others have searched for molecules that disrupt CD40-CD40L binding, no screen has been conducted in B cells to identify for CD40-specific inhibitors of NF-kB signaling - ours will be the first. In our revised application, we present new preliminary data that demonstrate our ability to perform counter screens that will identify small molecules that are specific to CD40-mediated signaling inside of human B cells. We have also revised Aim 3 to overcome the target ID problem inherent in pathway-based screens. Specific Aim 1: Collaborate with BIPDeC to implement a validated HTS (luciferase assay in our BL2- NFkB-luc cells stimulated with tCD40L) to identify inhibitors of the CD40 signaling pathway. We will use a cell-based, NF-kB luciferase assay to screen ~450,000 MLPCN probe compounds. Specific Aim 2: Employ established reagents to conduct secondary counter screens to validate that our hits are specific to the CD40 signaling pathway. To ensure that hit compounds are specific to intracellular CD40-mediated signaling, we will perform counter screens at the Broad Institute to (a) remove compounds that inhibit TNF? and LPS signaling, (b) remove compounds that interfere with the luciferase assay, and (c) remove compounds that are toxic to BL2 cells. We will test hit compounds in primary B-cells. Specific Aim 3: Apply SILAC (stable isotope labeling by amino acids in cell culture)-based target identification technology to identify the protein target from our drug screen. The approach is based on protein mass-spectrometry. We will apply SILAC to identify proteins that bind to small molecules identified in Aim 2. We will validate SILAC hits using a variety of molecular techniques, including Western blots, enzymatic assays and RNAi in cell lines and primary cells. IMPACT: We have preliminary data to demonstrate our ability to conduct all aspects of the study - including new data on counter screens and SILAC. Upon completion of our study, we will have identified small molecule inhibitors that are specific to CD40-mediated NF-kB signaling in human B cells.